中药材地骨皮PCR-RFLP鉴别方法研究  

Research on the Identification Method of Cortex lycii by PCR-RFLP

作　　者：王庆 董文静 张美 齐喜红 张宇欣 胡寒雪 Wang Qing;Dong Wenjing;Zhang Mei;Qi Xihong;Zhang Yuxin;Hu Hanxue(Ningxia Hui Autonomous Region Institute of Drug Inspection,Yinchuan 750002,China;College of Pharmacy,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏回族自治区药品检验研究院,银川750002 [2]宁夏医科大学药学院,银川750004

出　　处：《中国药事》2025年第3期317-326,共10页Chinese Pharmaceutical Affairs

基　　金：宁夏回族自治区重点研发计划项目(编号2023BEG03060);国家药品监督管理局药品监管科学体系建设重点项目(编号RS2024Z006)。

摘　　要：目的:建立中药材地骨皮聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)鉴别方法,有效区分地骨皮正伪品,保障其用药安全。方法:运用SnapGene软件对地骨皮正品来源中国枸杞(Lycium chinense)和宁夏枸杞(Lycium barbarum),伪品来源鹅绒藤(Cynanchum chinense)、黑果枸杞(Lycium ruthenicum)、香加皮(Periploca sepium)的ITS序列进行比对分析,筛选出差异限制性内切酶位点Eag I,利用Eag I对ITS序列PCR扩增产物进行酶切鉴别。主要对药材DNA提取方法,PCR扩增的退火温度、循环数及不同酶的扩增效果,酶切反应时间和酶切底物量进行优化,建立地骨皮PCR-RFLP鉴别方法,并对该方法的稳定性、准确性进行考察。结果:药材DNA适宜提取方法为试剂盒法,PCR扩增适宜退火温度为67℃、适宜循环数为35。Eag I酶切反应适宜底物量为25μL、温度为37℃、反应30min,地骨皮正品PCR扩增产物可被酶切产生酶切条带,而地骨皮伪品PCR扩增产物不能被酶切。结论:研究建立了地骨皮正伪品的PCR-RFLP鉴别方法,可为地骨皮市场流通鉴别提供技术支撑。Objective:To establish a polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)identification method for cortex lycii for effectively distinguishing between the authentic and fake products of cortex lycii and ensuring the safety of their medication.Methods:The ITS sequences of authentic medicinal herbs from Lycium chinense and Lycium barbarum,and fake products from Cynanchum chinense,Lycium ruthenicum and Periploca sepium were compared and analyzed with SnapGene software.The heterogeneous restriction endonuclease sites Eag I was selected,and PCR amplification products of their ITS sequences were identified using Eag I.The DNA extraction method for cortex lycii,annealing temperature,cycle number,and amplification efficiency of different enzymes for PCR amplification,as well as the enzyme digestion reaction time and substrate amount were optimized,and the PCR-RFLP identification method was established.On this basis,the stability and accuracy of this method were investigated.Results:The suitable extraction method for DNA from herbs slices was the reagent kit method. The suitable annealing temperatureand cycle number for PCR amplification were 67 ℃ and 35, respectively. The optimal substrate amount for theEag I restriction enzyme digestion reaction was 25 μL, and the suitable digestion temperature and reactiontime was 37 ℃ and 30 min, respectively. On the above conditions, the PCR amplification products of authenticmedicinal herbs were digested and displayed one band, however, the PCR amplification products of fakeproducts were not digested. Conclusion:The PCR-RFLP identification method for the authentic and fakeproducts of cortex lycii was established, and this method would provide technical support for the marketcirculation identification.

关 键 词：地骨皮 正品药材 伪品 聚合酶链式反应-限制性片段长度多态性 鉴别方法 

分 类 号：R917[医药卫生—药物分析学]